Bile secretion is a major function of the liver which is frequently impaired in diseases of the liver resulting in the syndrome of cholestasis. The long term objectives of this grant application, funded continuously since 1973, have been to characterize the basic transport mechanisms in hepatocytes that determine the secretion of bile and to define at the cellular and molecular level, alterations in these mechanisms that result in cholestatic liver disease. In this competitive renewal proposal the specific aims are #1. To understand the molecular mechanisms for adaptive regulation of basolateral hepatocyte membrane transporters (specifically MRP4 and OST1-OST2) in human liver that are important determinants of the adaptive response in cholestatic liver injury and to devise new therapies based on this information. In particular we will attempt to characterize the transcriptional regulators of the human MRP4 promoter as well as to continue to assess the role of OST2 in the adaptive regulation of the human heteromeric, facilitated bile salt transporter, OST1-OST2, and to determine the effect of bile duct ligation in the Ost1 knock out mice. In order to examine new therapeutic approaches we will assess the synergistic therapeutic effects of the combination of all-trans retinoic acid + ursodeoxycholic acid on human transporter gene expression in HepG2 cells and in animal models of cholestasis. In Aim # 2. we will examine the determinants by which the expression of the bile salt export pump (Bsep) is regulated in normal and cholestatic liver injury by focusing on the role of the ubiquitin/proteasome degradation pathway as a determinant of the regulation of surface expression of Bsep. Finally in Aim # 3. we will investigate the functional roles of proteins involved in maintaining canalicular apical membrane structural polarity. To do this we will determine the role of scaffolding proteins in the targeting and regulation of the ABC transporter, Mrp2. We will also assess the role of FIC1 in the regulation of human FXR expression and activity in human cell lines and rat hepatocytes by knocking down Fic1 using siRNA, as well as determining the role of Fic1 in maintaining canalicular structure and Bsep and Mrp2 function in rat collagen sandwich hepatocyte cultures. Public Health Relevance Narrative: This grant application supports research that focuses on understanding how the liver adapts to injury resulting from impairment of bile production (known as cholestasis). Bile formation is a vital function and its impairment in a variety of cholestatic liver diseases often results in progressive cholestasis and liver failure that can result in death or liver transplantation. By understanding the mechanism of adaptations of bile transporter proteins at the cellular and molecular level in cholestatic liver injury, we hope to design new therapeutic strategies that will augment these adaptive responses and retard or reverse the progression of these potentially fatal liver disorders.